Cardiac myosin binding protein-C (cMyBP-C) is a trans-filament protein which, at its N'-region, connects thick and thin filaments to regulate cardiac contractility. The overall objective of the studies contained in this renewal of R01HL-105826 is to define the N'-region of cMyBP-C as a critical regulator of cardiac contractility. In particular, C0-C1f, which interacts only with actin, is generated by proteolysis of cMyBP-C during ischemia/reperfusion (I/R) injury and heart failure. On the other hand, C0-C2 interacts with both actin and myosin, connecting thin and thick filaments to regulate sarcomere function. On the basis of these findings, we will 1) use a novel transgenic mouse model expressing cMyBP-C (cMyBP-C110kDa) in which the C0-C1f region was ablated to study the necessity and sufficiency of this domain in regulating cardiac contractility and 2) use adeno-associated virus 9 (AAV9)-mediated expression of (i) recombinant C0-C2 to determine the sufficiency of N'-terminal C0-C2 in bundling thick and thin filaments in vivo and (ii) recombinant C0-C2?CTS, in which the calpain-targeted site (CTS) has been ablated, to determine its therapeutic potential in improving cardiac function pre- and post-I/R injury. The molecular mechanisms underlying the regulation of cMyBP-C and, in turn, its impact on sarcomere structure and function, are largely unknown. Our short-term goal is to elucidate the specific role(s) of the N'-region of cMyBP-C in the regulation of cardiac function, whereas our long-term goal is to determine the mechanisms by which cMyBP-C stabilizes sarcomeric structure and function, thereby conferring cardioprotection during I/R injury. SPECIFIC AIM 1 will test the hypothesis that the C0-C1f domain of cMyBP-C, which is cleaved and released during I/R injury, is necessary for regulating cardiac function. Transgenic cMyBP-C110kDa mice will be used to determine the critical role of the N'-region of cMyBP-C at the sarcomere and whole-heart levels, compared to the control non-transgenic mice. SPECIFIC AIM 2 will test the hypothesis that the C0-C2 domains of cMyBP-C are sufficient to bundle thick and thin filaments and to regulate normal cardiac function. AAV9-mediated expression of recombinant C0-C2 will be used to prevent or rescue contractile dysfunction in mouse models that lack either the N'-region (C0-C1f) or full-length cMyBP-C in vivo. SPECIFIC AIM 3 will test the hypothesis that expression of recombinant C0-C2?CTS in vivo confers cardioprotection during I/R injury. Recombinant C0-C2?CTS protein is protected from calpain-mediated degradation. We expect AAV9-mediated expression of recombinant C0-C2?CTS to significantly reduce infarct size and apoptosis, as well as preserve contractile function during I/R injury. Together, these studies will determine the necessity and sufficiency of the N'-region (both C0-C1f and C0-C2) of cMyBP-C to regulate cardiac function, and, hence, provide therapy for myocardial injury and heart failure occurring during I/R injury.